Alveolar rhabdomyosarcoma (ARMS) is an aggressive pediatric cancer of the striated muscle lineage that is characterized by 2;13 or 1;13 chromosomal translocations. These events juxtapose PAX3 or PAX7 with FKHR to create fusion genes encoding PAX3-FKHR or PAX7-FKHR fusion products. The wild-type genes encode transcription factors, and the fusion products combine the PAX3 or PAX7 DNA binding domain and FKHR activation domain to create potent transcriptional activators. Though some initial studies indicated that these fusion proteins induce oncogenic effects, more recent studies have revealed genetic and phenotypic complexities that require refinement of the model for the role of these fusion products in ARMS tumorigenesis. These complexities are evidenced by gene transfer studies of these fusion genes that show oncogenic behavior in some conditions and growth suppressive behavior in other conditions. In addition, there are frequent alternative splicing events in the PAX3/PAX7-FKHR DNA binding domain that generate a mixture of functionally distinct isoforms. Finally, there are secondary genetic changes in ARMS cells, including gene amplification and small mutations, which may interact with the translocation events. To explain these findings, the hypothesis is proposed that the growth effects of these fusion proteins are initially dependent on the level of transcriptional activity, with oncogenic effects predominating at lower activity and growth suppression predominating at higher activity. The final balance between these growth effects as well as additional phenotypic activities is postulated to be modulated by other factors, including the mixture of functionally distinct isoforms and secondary genetic changes. The current proposal will focus on the PAX3-FKHR fusion protein and will explore these hypotheses by using inducible cell culture systems that display this range of growth effects to analyze the functional requirements and gene expression events associated with oncogenic effects and growth suppression. Cell culture and in vitro systems will be used to assess differences in DNA binding activity, phenotypic effects, and downstream expression events between the two PAX3-FKHR isoforms. Finally, these studies will investigate the role of amplified oncogenes and other genetic events in modifying the function and downstream effects of the fusion protein. These combined studies will permit a comprehensive evaluation of the relationship between PAX3-FKHR transcriptional function, target gene expression, phenotypic effects, and collaborating events, and will thereby better define the role of these fusion genes in ARMS tumorigenesis.